Xi Caiyun, Zhang Jianxi, Huang Zhifeng, He Liqiong, Zou Kailu, Xu Xiaoping, Guo Qulian, Sun Bei, Huang Changsheng
Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha 410008.
National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Changsha 410008, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2025 Apr 28;50(4):586-601. doi: 10.11817/j.issn.1672-7347.2025.240270.
Neuropathic pain (NP) is one of the most common forms of chronic pain, yet current treatment options are limited in effectiveness. Peripheral nerve injury activates spinal microglia, altering their inflammatory response and phagocytic functions, which contributes to the progression of NP. Most current research on NP focuses on microglial inflammation, with relatively little attention to their phagocytic function. Early growth response factor 2 (EGR2) has been shown to regulate microglial phagocytosis, but its specific role in NP remains unclear. This study aims to investigate how EGR2 modulates microglial phagocytosis and its involvement in NP, with the goal of identifying potential therapeutic targets.
Adult male Sprague-Dawley (SD) rats were used to establish a chronic constriction injury (CCI) model of the sciatic nerve. Pain behaviors were assessed on days 1, 3, 7, 10, and 14 post-surgery to confirm successful model induction. The temporal and spatial expression of EGR2 in the spinal cord was examined using real-time quantitative PCR (RT-qPCR), Western blotting, and immunofluorescence staining. Adeno-associated virus (AAV) was used to overexpress in the spinal cord, and behavioral assessments were performed to evaluate the effects of EGR2 modulation of NP. CCI and lipopolysaccharide (LPS) models were established in animals and microglial cell lines, respectively, and changes in phagocytic activity were measured using RT-qPCR and fluorescent latex bead uptake assays. After confirming the involvement of microglial phagocytosis in NP, AAV was used to overexpress in both in vivo and in vitro models, and phagocytic activity was further evaluated. Finally, eukaryotic transcriptome sequencing was conducted to screen differentially expressed mRNAs, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to identify potential downstream effectors of EGR2.
The CCI model successfully induced NP. Following CCI, EGR2 expression in the spinal cord was upregulated in parallel with NP development. Overexpression of EGR2 via spinal AAV injection enhanced microglial phagocytic activity and increased pain hypersensitivity in rats. Both animal and cellular models showed that CCI or LPS stimulation enhanced microglial phagocytosis, which was further amplified by overexpression. Transcriptomic analysis of spinal cord tissues from CCI rats overexpressing revealed upregulation of numerous genes associated with microglial phagocytosis and pain regulation. Among them, Lag3 emerged as a potential downstream target of EGR2.
EGR2 contributes to the maintenance of NP by enhancing microglial phagocytosis in the spinal dorsal horn.
神经性疼痛（NP）是慢性疼痛最常见的形式之一，但目前的治疗选择效果有限。外周神经损伤会激活脊髓小胶质细胞，改变其炎症反应和吞噬功能，这促使了NP的进展。目前大多数关于NP的研究集中在小胶质细胞炎症，而对其吞噬功能的关注相对较少。早期生长反应因子2（EGR2）已被证明可调节小胶质细胞的吞噬作用，但其在NP中的具体作用仍不清楚。本研究旨在探讨EGR2如何调节小胶质细胞的吞噬作用及其在NP中的作用，以确定潜在的治疗靶点。
成年雄性Sprague-Dawley（SD）大鼠用于建立坐骨神经慢性压迫损伤（CCI）模型。在术后第1、3、7、10和14天评估疼痛行为，以确认模型诱导成功。使用实时定量PCR（RT-qPCR）、蛋白质免疫印迹法和免疫荧光染色检测脊髓中EGR2的时空表达。腺相关病毒（AAV）用于在脊髓中过表达，进行行为评估以评价EGR2对NP的调节作用。分别在动物和小胶质细胞系中建立CCI和脂多糖（LPS）模型，使用RT-qPCR和荧光乳胶珠摄取试验测量吞噬活性的变化。在确认小胶质细胞吞噬作用参与NP后，AAV用于在体内和体外模型中过表达，进一步评估吞噬活性。最后，进行真核转录组测序以筛选差异表达的mRNA，随后进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路分析，以鉴定EGR2潜在的下游效应分子。
CCI模型成功诱导了NP。CCI后，脊髓中EGR2的表达与NP的发展同步上调。通过脊髓注射AAV过表达EGR2可增强小胶质细胞的吞噬活性，并增加大鼠的疼痛超敏反应。动物和细胞模型均显示，CCI或LPS刺激可增强小胶质细胞的吞噬作用，过表达EGR2可进一步放大这种作用。对过表达EGR2的CCI大鼠脊髓组织进行转录组分析，发现许多与小胶质细胞吞噬作用和疼痛调节相关的基因上调。其中，Lag3成为EGR2潜在的下游靶点。
EGR2通过增强脊髓背角小胶质细胞的吞噬作用，促进NP的维持。
